Project Summary Circadian rhythms are nearly ubiquitous endogenous timing systems that help coordinate the myriad physiological, metabolic and developmental processes that occur continuously in each organism at all times of the day. This circadian clock is comprised at the molecular level of interlocked and autoregulatory feedback loops that are built from complex interactions that are constantly changing in relation to each throughout the 24 hour cycle. The long term goal of this project is to understand the functional relationships among the inter- and intracellular processes that keep the circadian oscillator running and coordinated across the plant. We are using genetic, genomic, biochemical and cell biological tools and strategies of the model plant Arabidopsis to identify the molecules and mechanisms that regulate the transport of clock proteins between the cytosol and nucleus. We are focused in part on how post-translational modifications of clock proteins affect both their positional and temporal intracellular localization. Gatekeeping features of the intracellular environment, such as the nuclear pore (NP), are also addressed, using select NP mutants, and at the level of a single molecular species. We are applying for the first time in circadian studies single cell imaging techniques using a photoswitchable fluorescent protein to assess features of clock protein movement and turnover that will be applicable to non-plant circadian systems. Long-distance circadian signaling from shoot to root will be explored using select photoreceptor, kinase and glucose-signaling mutants as well as aberrant meristem mutants. Unbiased mutant screens to identify proteolytic factors controlling clock protein levels will help understand the importance of precise time-of-day phasing of these factors. We are also exploiting certain plant-specific advantages of small RNA processing to address other post-transcriptional control mechanisms of the circadian clock. Taken together our program will probe mechanisms of circadian control that should be broadly applicable across all eukaryotic systems.